ORIGINAL RESEARCHpublished: 10 April 2018

doi: 10.3389/fmicb.2018.00696

Frontiers in Microbiology | www.frontiersin.org 1 April 2018 | Volume 9 | Article 696

Edited by:

Peter Mullany,

University College London,

United Kingdom

Reviewed by:

Ariadnna Cruz-Córdova,

Hospital Infantil de México Federico

Gómez, Mexico

D. Ipek Kurtboke,

University of the Sunshine Coast,

Australia

Maria Soledad Ramirez,

California State University, Fullerton,

United States

*Correspondence:

Heejoon Myung

hjmyung@hufs.ac.kr

Specialty section:

This article was submitted to

Antimicrobials, Resistance and

Chemotherapy,

a section of the journal

Frontiers in Microbiology

Received: 07 November 2017

Accepted: 26 March 2018

Published: 10 April 2018

Citation:

Cha K, Oh HK, Jang JY, Jo Y,

Kim WK, Ha GU, Ko KS and Myung H

(2018) Characterization of Two Novel

Bacteriophages Infecting

Multidrug-Resistant (MDR)

Acinetobacter baumannii and

Evaluation of Their Therapeutic

Efficacy in Vivo.

Front. Microbiol. 9:696.

doi: 10.3389/fmicb.2018.00696

Characterization of Two NovelBacteriophages InfectingMultidrug-Resistant (MDR)Acinetobacter baumannii andEvaluation of Their TherapeuticEfficacy in Vivo

Kyoungeun Cha 1,2, Hynu K. Oh 1, Jae Y. Jang 1, Yunyeol Jo 1, Won K. Kim 1, Geon U. Ha 1,

Kwan S. Ko 3 and Heejoon Myung 1,2*

1Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies, Yong-In, South Korea, 2 The

Bacteriophage Bank of Korea, Hankuk University of Foreign Studies, Yong-In, South Korea, 3 Samsung Medical Center,

Sungkyukwan University School of Medicine, Suwon, South Korea

Acinetobacter baumannii is emerging as a challenging nosocomial pathogen due to

its rapid evolution of antibiotic resistance. We report characterization of two novel

bacteriophages, PBAB08 and PBAB25, infecting clinically isolated, multidrug-resistant

(MDR) A. baumannii strains. Both phages belonged to Myoviridae of Caudovirales as

their morphology observed under an electron microscope. Their genomes were double

stranded linear DNAs of 42,312 base pairs and 40,260 base pairs, respectively. The two

phages were distinct from known Acinetobacter phages when whole genome sequences

were compared. PBAB08 showed a 99% similarity with 57% sequence coverage to

phage AB1 and PBAB25 showed a 97% similarity with 78% sequence coverage to

phage IME_AB3. BLASTN significant alignment coverage of all other known phages were

<30%. Seventy six and seventy genes encoding putative phage proteins were found

in the genomes of PBAB08 and PBAB25, respectively. Their genomic organizations

and sequence similarities were consistent with the modular theory of phage evolution.

Therapeutic efficacy of a phage cocktail containing the two and other phages were

evaluated in a mice model with nasal infection of MDR A. baumannii. Mice treated with

the phage cocktail showed a 2.3-fold higher survival rate than those untreated in 7 days

post infection. In addition, 1/100 reduction of the number of A. baumannii in the lung of

the mice treated with the phage cocktail was observed. Also, inflammatory responses

of mice which were injected with the phage cocktail by intraperitoneal, intranasal, or oral

route was investigated. Increase in serum cytokine wasminimal regardless of the injection

route. A 20% increase in IgE production was seen in intraperitoneal injection route, but not

in other routes. Thus, the cocktail containing the two newly isolated phages could serve

as a potential candidate for therapeutic interventions to treat A. baummannii infections.

Keywords: multidrug-resistance, Acinetobacter baumannii, bacteriophage therapy, mouse model, genome

analysis

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

INTRODUCTION

Acinetobacter baumannii is an opportunistic pathogen causingnosocomial infection in hospitals. It is designated as anESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumonia, A. baumannii, Pseudomonas aeruginosa, and

Enterobacter species) pathogen by World Health Organization(WHO) (McConnell et al., 2013). It causes mainly pneumoniaand additionally burn infections, meningitis, urinary tractinfections, and sepsis (Dijkshoorn et al., 2007). Antibiotic-resistant strains are rapidly emerging and even multidrug-resistant (MDR) and pandrug-resistant (PDR) strains areobserved (Tuon et al., 2015). Its virulence factors include porins,lipopolysaccharides, capsular polysaccharides, metal acquisitionsystems, phospholipases, outer membrane vesicles, and proteinsecretion systems (Weber et al., 2016; Lee et al., 2017). Factorsinfluencing antibiotic resistance includes two-componentsystems AdeRS, BaeSR, GacSA, and PmrAB (Kröger et al., 2017).Treatment of carbapenem-resistant A. baumannii (CRAB)involves the use of combinations of last resort agents including

colistin and tigecycline, but the efficacy and safety issues are notcleared yet (Doi et al., 2015).

Phage therapy is a promising tool for controlling drug-resistant bacteria (Abedon, 2017; Lin et al., 2017). Themechanism of drug-resistance is totally unrelated to that ofphage infection. Although bacteriophages have been used as anantibacterial for almost 100 years, mainly in eastern European

countries, they are not recognized as drugs due to the lack ofa proper documentation needed for drug approval. In addition,we still wait for fulfillment of regulatory affairs to approve phagedrugs (Huys et al., 2013).

Novel therapies including bacteriophages against drug-resistant A. baumannii have been reported and reviewed (Mihuand Martinez, 2011; García-Quintanilla et al., 2013; Parasionet al., 2014). Successful phage control of various strains ofAcinetobacter was demonstrated not only in vitro but alsoin vivo. Two newly isolated phages infecting A. baumanniiwere characterized and suggested as potential candidates forphage cocktail (Merabishvili et al., 2014). Other two newlyisolated phages were characterized at genomic DNA level andsuggested as potential candidate for phage cocktail against CRAB(Jeon et al., 2016a). Phage B8-C62 was used to successfullycontrol CRAB infection via nasal route in mice model (Jeonet al., 2016b). Phage vB-GEC_Ab-M-G7 was used to successfullycontrol wound infection in a rat model (Kusradze et al., 2016).Medes et al. tested phage cocktails against diabetic cutaneouswounds in two animal models. Phage cocktails for Staphylococcusaureus or Pseudomonas aeruginosa improved wound healing, butcocktail for A. baumannii was not as effective. A personalizedphage cocktail for treating mouse dorsal wound model wasreported (Regeimbal et al., 2016). Phages from multi-institutelibraries were used to make a personalized cocktail and proveneffective for treating a diabetic human patient with necrotizingpancreatitis complicated by an MDR A. baumannii infection(Schooley et al., 2017). A phage was effective in resolvingwound infection caused by multidrug-resistant A. baumannii inan uncontrolled diabetic rat model (Shivaswamy et al., 2015).

A bacteriophage-containing aerosol was proven effective forcleaning and decreased the rates of infection caused by CRABin intensive care units (Wang et al., 2016). A combined lysisspectrum of four lytic phages against clinically isolated CRABwas reported to be 87.5% and phages were proven effective astherapeutic agents for lung infection without deleterious sideeffects in mice model (Hua et al., 2018).

Here, we report bothmicrobiological characterization of novelphages infectingA. baumannii and the efficacy of a phage cocktailcontaining the phages in control of nasal infection in a micemodel. Further, we provide the basis why the phages can be usedas a therapeutic intervention practically.

MATERIALS AND METHODS

Ethics StatementAll animal studies were approved by and followed theguidelines and regulations of the Ethical Committee for AnimalExperiments of Hankuk University of Foreign Studies (approvalnumber 2017-0001).

Bacterial Strains and Phages UsedFourteen clinical A. baumannii strains were obtained frompatients in Samsung Medical Center, Sungkyukwan UniversitySchool of Medicine. Antibiotic resistance profile of the strainsis shown in Table 1. To determine the genotypes of theA. baumannii isolates, Oxford scheme multilocus sequencetyping (MLST) was performed as described previously (Bartualet al., 2005; http://pubmlst.org/abaumannii/info/primers_Oxford.shtml). One of the strains (strain 28) was selected andfurther used for mice experiment. It was resistant to bothkanamycin (3.5 mg/ml, Sigma, USA) and ampicillin (5 mg/ml,Sigma, USA), and this fact was used to enumerate recoveredbacteria from mice lung. The 9 bacteriophages tested against the14 strains were PBAB05, PBAB06, PBAB07, PBAB08, PBAB25,PBAB68, PBAB80, PBAB87, and PBAB93. Bacteriophagesused for making the therapeutic cocktail were PBAB08,PBAB25, PBAB68, PBAB80, PBAB93 (Bacteriophage Bank ofKorea).

Phage Isolation and PurificationBacteriophage isolation and characterization were describedthoroughly (Clokie and Kropinski, 2009a,b; Clokie et al., 2018).The isolation and characterization of phages have been describedpreviously (Kim et al., 2013). The phages were purified usinga glycerol gradient centrifugation method (Sambrook andRussell, 2006). A single plaque was used to inoculate 5mlof a mid-exponential-phase culture of the bacterium, followedby incubation at 37◦C for 3 h. A phage lysate was obtainedby centrifugation at 11,000 xg for 10min and discarding thesupernatant. Five ml of the lysate was used to inoculate 100mlof a mid-exponential-phase culture of the bacterium, and themixture was then incubated until lysis was completed. NaClwas added to the lysate at a final concentration of 1M, and itwas incubated at 4◦C for 1 h. After centrifugation at 11,000x gfor 10min, 10% (wt/vol) polyethylene glycol 8000 (PEG 8000)was added, and the mixture was then incubated at 4◦C for 1 h.

Frontiers in Microbiology | www.frontiersin.org 2 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

TABLE 1 | Antibiotic resistance profile of clinically isolated Acinetobacter baumannii strains and their phage susceptibility.

Antibiotics/

name of isolate Sequencetype(ST)

Imipenem

Meropenem

Ampicillin/sulbactam

Ceftazidim

e

Cefepim

e

Gentamicin

Ciprofloxacin

Levofloxacin

Trimethoprim/

Sulfamethoxazole

Piperacillin/tazobac

colistin

Tigecycline

PBAB number* of

infecting phages

Strain 21 191 >64** >64 >64 >64 >64 >64 >64 64 >64 >256 1 8 none

Strain 32-a 191 >64 >64 >64 >64 >64 >64 >64 >64 >64 >256 1 8 none

Strain F-224 1240 >64 >64 >64 >64 >64 >64 >64 >32 >64 >256 1 8 5, 6, 7, 87

Strain F-1208 357 >64 >64 >64 >64 >64 >64 >64 32 >64 >256 >64 1 8, 68, 80, 93

Strain F-1510 191 >64 >64 >64 >64 >64 >64 >64 16 >64 >256 2 8 none

Strain F-1629 357 >64 >64 32 >64 64 >64 >64 32 >64 >256 >64 8 8, 68, 80, 93

Strain 26 368 16 16 32 64 64 16 4 32 32 128 64 4 8, 68, 80,

Strain 28 368 16 16 16 64 64 16 4 32 32 128 1 8 8, 25, 68, 80, 93

Strain 32-b 357 16 16 16 64 64 16 4 32 32 128 4 2 8, 68, 80, 93

Strain 54 208 16 16 32 64 64 16 4 8 32 128 1 2 none

Strain 58 208 16 16 32 64 64 16 4 8 32 128 1 2 none

Strain 81 191 16 16 32 64 64 16 4 8 32 128 4 4 5, 6, 7, 87

Strain K20-B-667 191 >64 >64 >64 >64 >64 >64 >64 >64 >64 >256 1 8 none

Strain K20-B-890 191 8 64 >64 32 64 >64 >64 >64 >64 256 1 32 none

*PBAB numbers according to the Bacteriophage Bank of Korea (www.phagebank.or.kr).

**The numbers in each box shows the maximum concentration of each antibiotic to which tested bacteria was resistant (mg/L).

Darkly shaded box means “resistant”, lightly shaded box “intermediate”, and white box “susceptible”.

The supernatant was discarded after centrifugation at 11,000xg for 10min. Then the pellet was resuspended in 750 µl ofSM buffer [100mM NaCl, 8mM MgSO4·7H2O, 50mM Tris-Cl(pH 7.5)], and chloroform was added at a ratio of 1:1 (vol/vol),followed by vortexing and then centrifugation at 3,000x g for15min. The upper phase was isolated and was added to apolycarbonate centrifuge tube containing 3ml of 40% glycerolin the lower layer and 4ml of 5% glycerol in the upper layer.After centrifugation at 151,000x g for 1 h, the supernatant wasdiscarded, and the pellet was resuspended in 400 µl of SM buffer.For animal experiments, any contaminating lipopolysaccharide(LPS) was removed before treatment. Triton X-114 (Sigma, USA)was added to phage solution at a final concentration of 1%(vol/vol) and mixed using vortex for 10 s. After incubation on icefor 10min, the mixture was transferred to 37◦C water bath andincubated for 1min. After a centrifugation at 15,000x g for 5min,supernatant was collected and used as the final phage solution.Purified phages were stored at 4◦C until use. Standard doubleagar overlay plaque technique was used for phage enumeration(Kropinski et al., 2009).

Transmission Electron Microscopy (TEM)of Phage ParticlesPurified phage sample was loaded onto a copper grid for 1minfollowed by negative staining with 2% (vol/vol) uranyl acetate(pH 6.7) and drying. The phage morphology was observedusing a Carl Zeiss LIBRA 120 EF-TEM (Carl Zeiss, Oberkochen,Germany) at an accelerating voltage of 120 kV.

Genome Sequencing, Annotation, andAnalysisWhole genome sequencing of phage DNA was carried outusing PacBio RS II system in Macrogen, Korea. Open readingframes (ORFs) were searched using NCBI ORF Finder. Genomicsequence similarity comparison was done using MAUVE. ORFmap was drawn using CLC Genomics Workbench 10. Genomictree was drawn using Mega 7.

One Step MultiplicationOne step growth experiment was described previously (Hymanand Abedon, 2009). Briefly, phage PBAB08 or PBAB25 was addedto a fresh culture of A. baumannii at an MOI 0.001 and allowedto adsorb for 5min. The mixture was then centrifuged at 1,738xg for 10min. The supernatant was discarded to remove any freephages and the pellet was resuspended in 3ml of fresh LB brothincubated at 37◦C with shaking. One hundred micro liters ofsample was removed from the culture every 5min and subjectedto titration using double-layer agar plate methods (Kropinskiet al., 2009). This assay was performed at least in triplicate.

Phage StabilityFor temperature stability test, phage titer was measured afterincubation of phage lysates for 1 h at different temperatures (4,37, 45, 55, 60, 65, or 80◦C) using a double-layer agar platemethod (Kropinski et al., 2009). For pH stability test, phage titerwas measured after 1 h’s incubation of phage lysates at roomtemperature by mixing with equal volume of pH buffer solutionsof different pH values (pH 3, 4, 5, 6, 7, 8, 9, 10, and 11). pH

Frontiers in Microbiology | www.frontiersin.org 3 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

buffer solutions were made by adding HCl drop by drop to 1MNaOH solution. The resulting pHwasmeasured using S220 sevencompactTM pH/Ion (Mettler Toledo, Columbus, USA).

Phage Protection Studies Using a MiceModelSix week-old female Balb/c mice were obtained from Young Bio,Korea. Mice were divided into 4 groups; Group 1 was treated withSM buffer only. Group 2 was infected with MDR A. baumannii(strain 28) only. Group 3 was treated with the phage cocktailonly. Group 4 was infected with A. baumannii and treated withthe phage cocktail. Each group contained 20 mice. Mice wereintraperitoneally injected with cyclophosphamide (Sigma, USA)at the concentration of 150 mg/kg at−1 and−3 days of bacterialinfection. Before intranasal bacterial or phage injection, micewere anesthetized with 125 mg/kg of avertin (Sigma, USA) byintraperitoneal injection. 1 × 108 CFU of A. baumannii wasintranasally injected to each mouse at 0 and +1 days. SM bufferwas injected to the control group mice instead. 1 × 109 PFUof phage cocktail was intranasally injected to experimental miceevery day from −1 to +7 days. At days 0 and +1, phageswere injected 4 h after bacterial injection. Survival of mice wasobserved until 7 days after bacterial infection. Bacterial load inmice lung was counted as follows; lung was isolated at +3 and+4 days and weight was measured. After homogenization, 500µl of SM buffer was added and was subjected to centrifugation at2,000x g. The supernatant was used for counting A. baumanniiin a medium containing both kanamycin (3.5 mg/ml) andampicillin (5 mg/ml).

Cytokine, IgE, and Histamin AssaysTo see immune reactions against phages, the cocktail wasintroduced every day for 7 days to mice in three different routes;intraperitoneal, intranasal, and oral. Three mice were used foreach route. Control group was treated with SM buffer. One dayafter the last treatment of a phage cocktail, mice were sacrificedand serum was obtained for analysis of cytokine profile usingMulti-Analyte ELISArray kits (Qiagen, USA), of IgE profile usingRayBio R© Mouse IgE ELISA Kit (RayBiotech, USA), and ofhistamine using Histamine Enzyme Immunoassay Kit (SPI Bio,France).

Statistical AnalysesStudent’s t-test (Bailey, 1959) was used to calculate thesignificance of the difference among test groups. For eachassay, all determinations were carried out at least in triplicate.Statistically significant values were defined as P < 0.05 orP < 0.01.

RESULTS

Antibiotic Resistance of Clinically IsolatedA. baumannii StrainsFourteen bacterial isolates were selected and their antibioticresistance and phage susceptibility were observed (Table 1).All the isolates were multidrug-resistant (MDR) strains. Manyof them were resistant even to colistin or tigecycline. The

14 A. baumannii isolates showed five sequence types (STs)based on MLST (Table 1), and they belonged to the sameclonal complex (CC), global complex II. Nine Acinetobacterbacteriophages, PBAB05, PBAB06, PBAB07, PBAB08, PBAB25,PBAB68, PBAB80, PBAB87, and PBAB93, were selected fromthe Bacteriophage Bank of Korea and tested for infection to theclinical A. baumannii strains. Seven out of 14 isolates could beinfected by four or five phages. The phage susceptibility was notrelated to antibiotic resistance profile. Isolate 28 was susceptibleto all five phages and thus selected for mice infection experimentslater. We further characterized two of the phages PBAB08and PBAB25 from which whole genomic DNA sequences weresuccessfully obtained as single contigs after next generationsequencing.

Bacteriophage CharacterizationTwo phages, PBAB08 and PBAB25, belonged to familyMyoviridae of order Caudovirales (Figures 1A,B). Their virionswere composed of a spherical head and a long, rigid tail. PhagePBAB08 had a head of 180 nm in diameter and a tail of 360 nmin length. The smaller phage PBAB25 had a head of 80 nm indiameter and a tail of 90 nm in length. A complete lysis of hostbacteria infected with PBAB08 or PBAB25 occurred in 25minpost infection (Figure 1C). Burst size for each phage was 215 and630, respectively.

The infectivity of PBAB08 and PBAB25 remained intact whenexposed to pHs ranging from 5 to 10 for 1 h (Figures 2A,B). Itdecreased rapidly at pHs above 10. The infectivity of both phagesremained intact when exposed to temperatures ranging from 4 to55◦C for 1 h and dropped rapidly above 65◦C (Figures 2C,D).

Genomic Sequence AnalysisThe whole genomes of phages PBAB08 and PBAB25 weresequenced (GenBank accession numbers MG366114 andMG366115, respectively). Their genomes were double strandedlinear DNAs of 42,312 base pairs and 40,260 base pairs,respectively. The genome sequences were compared to reportedAcinetobacter phages using BLASTN. PBAB08 showed a 99%similarity with 57% sequence coverage to phage AB1 (Yanget al., 2010), and PBAB25 showed a 97% similarity with 78%sequence coverage to phage IME_AB3 (Zhang et al., 2015).BLASTN significant alignment coverage of all other knownphages were <30%. A mosaic structure between the genomesof PBAB08 and AB1, and that of PBAB25 and IME_AB3 wereobserved (Figure 3). An extensive rearrangement occurredbetween genomes of PBAB08 and AB1, while genomic structurewas conserved between PBAB25 and IME_AB3.

Seventy six and seventy genes encoding phage proteins werefound in the genomes of PBAB08 and PBAB25, respectively(Figure 4). Functional annotation of putative ORFs of each phageis shown in Tables 2, 3. They are divided into four categoriesaccording to functions; structural proteins, those involved inDNA packaging, those involved in replication and regulation,and those involved in lysis. It was evident that sequencesimilarities for both pairs weremore prominent in virion proteinsthan in proteins involved in replication and regulation. It isnot fair to compare virion proteins of the two pairs in parallel

Frontiers in Microbiology | www.frontiersin.org 4 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

FIGURE 1 | Transmission electron micrographs of bacteriophages PBAB08 (A) and PBAB25 (B). Samples were negatively stained with uranyl acetate. Scale bar is

shown in each picture. (C) One step multiplication of phages PBAB08 (solid line) and PBAB25 (broken line).

since the extent of annotations were different. Nevertheless,it is notable that PBAB25 had a wide array of tail proteinssuggesting a complicated tail structure. Since both phages hadtheir own putative DNA polymerases, a phylogenetic tree ofclosely related Acinetobacter phages in GenBank was drawnbased on the sequence comparison of their DNA polymerasegenes (Figure 5). The phages could be grouped as two, in whichPBAB08 belonged to one group (upper 10 phages in Figure 5),while PBAB25 belonged to the other group (lower 10 phages). NoDNA polymerase was found in functional annotation of ORFs ofphage AB1, thus it was not included in the tree.

In Vivo Efficacy of Phage CocktailOnly 15% of mice infected with A. baumannii intranasallysurvived at 7 days post infection (Figure 6A). On the contrary,35% ofmice infected with the bacteria followed by treatment withthe phage cocktail survived. A better survival of phage-treatedmice was observed at day 4 post infection. Sixty percentage ofmice survived with phage treatment while 20% of mice survivedwith only bacterial infection. Mice treated with phage onlyremained healthy for the entire experimental period.

Reduction of bacterial load in infected lung of mice wasobserved (Figure 6B). At 3 and 4 days post bacterial infection,bacteria resistant to both kanamycin and ampicillin werecounted. Double-resistant bacterial count was reducedmore than100-fold for both days. Nevertheless, it was clear that a complete

elimination of A. baumannii was not achieved with this phagetreatment.

Immune Reactions Against the PhageCocktailPhages are foreign substances to mice and there are chances theycould elicit immune responses.We checked changes in serum IgElevel indicating any allergic responses for three different routes ofphage injection (Figure 7A). A 20% increase in serum IgE wasobserved for intraperitoneal injection route, while no significantchange was observed for either intranasal or oral administration.For cytokines, a slight increase of serum GM-CSF was observedfor all three routes of phage administration (Figure 7B). Inintraperitoneal route, slight increases of IL2, IL10, and IL17Awere also observed. Thus, inflammatory response against thephage cocktail was minimal.

DISCUSSION

Among the clinically isolated MDR A. baumannii strains usedin this study, some of them were infected by three or morephages, while the others were not infected by any of the fivephages used. The degree of drug-resistance of a host bacteriumwas not related to the number of phages infecting the samehost, indicating that acquirement of drug resistance, probably byhorizontal gene transfer (Huang et al., 2015; Krahn et al., 2016;

Frontiers in Microbiology | www.frontiersin.org 5 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

FIGURE 2 | Stability of phages at various pHs and temperatures. Remaining infectivity of phages PBAB08 (A) and PBAB25 (B) were measured after exposure of

phages to indicated pHs for 1 h. Remaining infectivity of phages PBAB08 (C) and PBAB25 (D) were measured after exposure of phages to indicated temperatures for

1 h. The experiments were carried out in a triplicate.

FIGURE 3 | Genomic sequence comparison between phages PBAB08, AB1, PBAB25, and IME_AB3 using MAUVE. Line-connected colored boxes indicate regions

of sequence similarity in corresponding phage genomes.

Frontiers in Microbiology | www.frontiersin.org 6 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

FIGURE 4 | Putative open reading frame (ORF) map of phages PBAB08 (A) and PBAB25 (B). ORF map was drawn using CLC Genomics Workbench 10. Each arrow

is color-coded according to annotated genomic function.

Zhang et al., 2017) did not involve alteration of phage receptor orreplication mechanisms. It further suggested that MDR bacteriacould be treated with phages as long as infecting phages could beisolated.

Head size of PBAB08 (180 nm) was twice that of PBAB25(80 nm). Yet, the lengths of genomic DNAs of the two phageswere about the same. For example, the genome size of phage T4 is169 kilobase pair and the head size varies between 85 and 120 nm(Keller et al., 1988). Although a minimal requirement should beenough room for containing entire phage genomic DNA, it seemsthat there are other determinants of the head size of a phage. Inthe case of T4, head size determinant included scaffolding coreand the shell of the procapsid, mutants of which resulted in variedhead sizes (Keller et al., 1988).

From pH and temperature stability data, phage infectivityshould remain intact at temperatures and pHs reaching welloutside normal human physiological conditions. This should bea good characteristics if the phages are intended for therapeuticapplications. A phage preparation in a suitable buffer kept in arefrigerator could remain stable until it encounters target bacteriawhen applied in vivo.

A mosaic in genomic structure observed from closely relatedphages in this study is consistent with the modular theoryof phage evolution where phage genomes are mosaics ofmodules that recombines freely in genetic exchanges involvingdifferent phages (Botstein, 1980). It was also observed in otherviruses including P2-like phages (Davies and Lee, 2006) andeven animal viruses (Botstein, 1980). It is no wonder that

viruses evolve rapidly by exchanging genetic materials andextend their diversity. The ease of such exchange in a hostcell could be one reason why phages are the most divergentbiological entities on earth. In the case of phage AB1 whichlacks its own DNA polymerase gene, the phage should dependon host DNA polymerase for its own replication. Still itsgenome retains genomic mosaic to PBAB08, suggesting thatmodular recombination among phage genomes was independentof their replication strategy. It should be noted that DNApolymerases of PBAB25 and phage Presley was closest insequence, but overall similarity between their genomic sequenceswas <30%. It suggests that multiple rounds of recombinationevents has occurred among various strains of phages during theevolution.

It is noteworthy that only 3 genes encoding putative tailproteins in PBAB08 with a longer tail was annotated while14 genes annotated in PBAB25 with much shorter tail. Itseems that tail length is not related to complexity of itsstructure. Although electron microscopy of phage IME_AB3,closest to PBAB25 in genomic DNA sequence, was notclear enough to tell the tail structure (Zhang et al., 2015),the two closely related phages of IME_AB3, Acinetobacterphage vB_AbaS_Loki (Turner et al., 2017) and Achromobacterphage phiAxp-1 (Li et al., 2016), have noncontractile tailsand are classified as Siphoviridae, while PBAB25 belongedto Myoviridae. Thus it suggests that sequence similarityof tail components does not ensure the similarity in tailmorphology.

Frontiers in Microbiology | www.frontiersin.org 7 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

TABLE 2 | Functional annotation of putative ORFs found in bacteriophage PBAB08.

ORF Function Related bacteria or phage Putative encoded phage protein Similarity (%)

9 Structural protein Acinetobacter phage WCHABP5 Putative internal virion protein B 100

11 Acinetobacter phage vB_AbaP_PD-6A3 Putative internal virion core protein 91

33 Acinetobacter phage phiAB6 Putative internal virion protein 75

46 Acinetobacter phage AB3 Tail tubular protein B 100

56 Acinetobacter phage YMC11/12/R2315 Putative head protein 92

61 Acinetobacter phage phiAC-1 Putative capsid protein 95

66 Acinetobacter phage phiAC-1 Putative tail fiber protein 88

68 Acinetobacter phage AB3 Putative internal virion protein B 73

69 Acinetobacter phage AB3 Putative internal virion protein B 97

73 Acinetobacter phage IME-AB2 Putative phage head portal protein 93

79 Acinetobacter bacteriophage AP22 Putative portal protein 91

86 Acinetobacter phage phiAC-1 Putative capsid protein 95

99 Acinetobacter phage IME-AB2 Putative phage head portal protein 95

100 Acinetobacter phage YMC-13-01-C62 Putative portal protein 98

111 Acinetobacter phage YMC-13-01-C62 Putative portal protein 90

114 Acinetobacter baumannii 99063 Putative membrane protein 94

121 Acinetobacter phage WCHABP12 Putative tail fiber protein 75

54 DNA packaging Acinetobacter phage IME-AB2 Putative phage terminase large subunit 98

78 Acinetobacter phage IME-AB2 Putative phage terminase large subunit 93

110 Acinetobacter phage IME-AB2 Putative phage terminase large subunit 90

2 Replication and

Regulation

Harpegnathos saltator Apolipoprotein D 77

7 Acinetobacter phage vB_AbaM_IME200 DNA polymerase I 73

16 Stenotrophomonas maltophilia Adenine phosphoribosyltransferase (fragment) 63

20 Acinetobacter phage vB_AbaM_IME200 Carboxypeptidase 95

24 Massilia sp. JS1662 Amino acid transporter 50

29 Staphylococcus aureus subsp. aureus Nickel ABC transporter, nickel/metallophore periplasmic

binding domain protein

89

40 Rathayibacter sp. VKM Ac-2630 Transcriptional regulator 75

52 Burkholderia sp. WSM4176 TonB-dependent receptor 60

109 Acinetobacter phage WCHABP12 Global DNA-binding transcriptional dual regulator 87

116 Acinetobacter phage YMC-13-01-C62 Putative RNA polymerase 92

119 Acinetobacter phage YMC-13-01-C62 Putative baseplate assembly protein 98

120 Acinetobacter phage WCHABP1 Baseplate J-like protein 98

125 Acinetobacter phage WCHABP1 Putative RNA polymerase 89

19 Lysis Acinetobacter phage WCHABP5 Putative holin 51

74 Acinetobacter phage WCHABP5 Putative holin 83

More than two-fold increase of survival from mice treatedwith phage cocktail was shown in this study. Although itproved a limited effectiveness, there could be a room forimprovements. In a previous report, mice survival after phagetreatment in a nasal infection model was higher than thisexperiment (Jeon et al., 2016b). But it should be noted that inthe same report mice were inoculated with phages only after30min of bacterial infection, in which bacterial proliferationtime was much shorter before confronting phages. In this study,there was a 4 h gap between bacterial infection and phageinoculation. In another report, open wound in rats were treatedwith phages after A. baumannii infection (Kusradze et al., 2016).Phage treatment was done at 12 h after bacterial infection, but

showed a higher reduction of bacterial load than this study.Depending on types of infection and routes of inoculation, theoutcome seems to vary. A pretreatment of phages in this studydid not help efficiently eliminating incoming bacteria. Mostof the phages could be destabilized and lost their infectivitybefore bacterial infection. In a wound infection model, loss ofphages during application would be less than nasal infectionmodel since there would be less chance of immune-mediatedclearance. Also, the amount of phages applied seems to becritical. A dose dependency was observed in a previous report(Jeon et al., 2016b).

Increase in serum proinflammaotry cytokines after phagetreatment, if any, could reflect a possible inflammatory response

Frontiers in Microbiology | www.frontiersin.org 8 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

TABLE 3 | Functional annotation of putative ORFs found in PBAB25.

ORF Function Relative bacteria or phage Putative encoded phage protein Similarity

46 Structural protein Acinetobacter phage vB_AbaM_ME3 Baseplate hub protein 97

67 Acinetobacter phage vB_AbaM_ME3 Head completion protein 100

77 Acinetobacter phage vB_AbaM_ME3 Baseplate hub protein 100

106 Acinetobacter phage IME_AB3 Putative portal protein 100

107 Acinetobacter phage IME_AB3 Putative scaffold protein 91

109 unclassified Siphoviridae Putative tail terminator protein 72

110 Acinetobacter phage IME_AB3 Putative major tail tube protein 98

112 Acinetobacter phage IME_AB3 Putative tail chaperonin protein 100

113 Acinetobacter phage IME_AB3 Putative tail tape measure protein 87

114 unclassified Siphoviridae Putative distal tail protein 86

116 Acinetobacter phage IME_AB3 Putative capsid and scaffold protein 100

135 Acinetobacter phage IME_AB3 Putative head protein 99

136 Acinetobacter phage IME_AB3 Putative major capsid protein 100

138 Acinetobacter phage IME_AB3 Putative structural protein 95

141 Acinetobacter phage IME_AB3 Putative tail tape measure protein 94

142 unclassified Siphoviridae Putative distal tail protein 86

144 Acinetobacter phage IME_AB3 Putative tail protein 91

167 Acinetobacter phage IME_AB3 Putative portal protein 100

168 Acinetobacter phage IME_AB3 Putative head protein 98

171 Acinetobacter phage IME_AB3 Putative major tail tube protein 98

172 Acinetobacter phage IME_AB3 Putative tail completion protein 100

173 Acinetobacter phage IME_AB3 Putative tail tape measure protein 67

174 Acinetobacter phage IME_AB3 Putative tail tape measure protein 100

175 Acinetobacter phage IME_AB3 Putative capsid and scaffold protein 99

176 Acinetobacter phage IME_AB3 Putative capsid and scaffold protein 100

177 Acinetobacter phage IME_AB3 Putative tail protein 100

178 Acinetobacter phage IME_AB3 Putative tail protein 99

179 Acinetobacter phage IME_AB3 Putative tail protein 90

184 Acinetobacter phage vB_AbaM_ME3 Putative membrane protein 98

38 DNA packaging Acinetobacter phage IME_AB3 Putative RecB exonuclease 100

101 Acinetobacter phage IME_AB3 Putative terminase small subunit 97

103 Acinetobacter phage IME_AB3 Putative terminase large subunit 96

128 Acinetobacter phage IME_AB3 Putative endonuclease 81

133 Acinetobacter phage IME_AB3 Putative terminase large subunit 97

166 Acinetobacter phage IME_AB3 Putative terminase small subunit 94

5 Replication and regulation Acinetobacter phage IME_AB3 Putative replicative primase/helicase 97

9 Acinetobacter phage IME_AB3 Putative single-stranded DNA-binding protein 100

10 Acinetobacter phage IME_AB3 Putative DNA/RNA helicase protein 84

11 Acinetobacter phage IME_AB3 Putative DNA polymerase subunit 95

36 Acinetobacter phage IME_AB3 Putative replicative primase/helicase 99

37 Acinetobacter phage IME_AB3 Putative MazG pyrophosphatase 88

39 Acinetobacter phage IME_AB3 Putative DNA/RNA helicase protein 42

40 Acinetobacter phage IME_AB3 Putative DNA polymerase subunit 99

71 Acinetobacter phage IME_AB3 Putative replicative primase/helicase 100

73 Acinetobacter phage vB_AbaM_ME3 Putative DNA polymerase subunit 100

74 Acinetobacter phage IME_AB3 Putative DNA polymerase subunit 84

121 Acinetobacter phage vB_AbaM_ME3 Adenine/guanine phosphoribosyltransferase 97

147 Acinetobacter phage vB_AbaM_ME3 Chemical-damaging agent resistance protein C 94

131 Lysis unclassified Siphoviridae Putative holin, class II 78

132 Acinetobacter phage IME_AB3 Putative endolysin 99

165 Acinetobacter phage IME_AB3 putative endolysin 99

Frontiers in Microbiology | www.frontiersin.org 9 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

FIGURE 5 | Genomic tree of Acinetobacter phages found in GenBank. The tree was drawn based on each phage’s DNA polymerase gene sequence using Mega 7.

FIGURE 6 | In vivo efficacy of the phage cocktail containing PBAB08 and PBAB25 in a mice nasal infection model. (A) Survival of 4 groups of mice observed for 7

days after bacterial infection. Group 1 (closed circle) was inoculated with SM buffer only. Group 2 (open circle) was infected with MDR A. baumannii strain 28 only.

Group 3 (triangle) was treated with the phage cocktail only. Group 4 (rectangle) was infected with A. baumannii and treated with the phage cocktail. (B) Bacterial load

in lungs of infected mice in 3 and 4 days post-infection with or without phage treatment. **P < 0.01.

elicited by phages. But it was minimal in this study. Miceremained healthy and behaved normally. The observation iscoherent with the previous finding that oral inoculation of T7phages induced a minimal increase in cytokine production ofmice (Park et al., 2014). Another phage, SH-Ab 15519, wasreported and the therapeutic efficacy was better than the phagecocktail used in this study (Hua et al., 2018). Applicationof only a single phage showed 90% survival of mice withthe M.O.I of 1 or 10. BLAST search revealed 24% coverage

with 92% identity when compared to PBAB08, meaning onlya low degree of relatedness. As in this study, application ofSG-Ab 15519 was generally safe based on histopathologicalexamination of mice lung treated with the phage and cytokineanalysis.

Taken together, bacteriophage treatment for mice nasallyinfected with clinically isolated MDR A. baumannii strainwas proven effective and safe. Depending on bacterial strainsand phages, an optimal condition for an effective treatment

Frontiers in Microbiology | www.frontiersin.org 10 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

FIGURE 7 | Observation of immune responses against phage treatment in mice. (A) Changes in IgE production after treatment of phages in three different routes.

(B) Changes in cytokine production after phage treatment in intranasal, oral, or intraperitoneal route. *P < 0.05, **P < 0.01.

needs to be set. Parameters should include selection of phagesfrom a bank collection based on susceptibility of the causativebacterial strain, composition of a cocktail containing differentphage-resistant group members, doses and lengths of phageapplications, and routes of phage application. A rapid inoculationof phages after exposure to the pathogen is another criticalrequirement.

Based on findings that the phage cocktail containingPBAB08 and PBAB25 is effective and safe for treatinginfections by MDR A. baumannii in vivo, phage therapywould be a viable alternative for antibiotics, especiallyin cases where antibiotics are not treatment options anymore.

AUTHOR CONTRIBUTIONS

HMandKK: designed experiments; KC, YJ,WK, and GH: carriedout experiments; HO and JJ: analyzed experimental results; HM:wrote the manuscript.

FUNDING

This work was supported by National Research Foundation(NRF) of Korea Funds 2015R1A2A2A01003632 and2017M3A9B8069292, ATC Program 10076996 from Ministry ofTrade, Industry, and Energy of Korea, and HUFS Research Fundof 2017.

REFERENCES

Abedon, S. T. (2017). Bacteriophage clinical use as antibacterial

“drugs”: utility and precedent. Microbiol. Spectr. 5:BAD-0003-2016.

doi: 10.1128/microbiolspec.BAD-0003-2016

Bailey, N. T. J. (1959). Statistical Methods in Biology. London: The English

Universities Press Ltd.

Bartual, S. G., Seifert, H., Hippler, C., Luzon, M. A., Wisplinghoff, H., and

Rodríguez-Valera, F. (2005). Development of a multilocus sequence typing

scheme for characterization of clinical isolates of Acinetobacter baumannii. J.

Clin. Microbiol. 43, 4382–4390. doi: 10.1128/JCM.43.9.4382-4390.2005

Botstein, D. (1980). A theory of modular evolution for bacteriophages. Ann. N.Y.

Acad. Sci. 354, 484–490.

Clokie, M. R. J., Kropinski, A.M., and Lavigne, R. (2018). “Bacteriophages methods

and protocols,” in Methods in Molecular Biology, Vol. 3 (New York, NY:

Springer Nature).

Clokie, M. R. J., and Kropinski, A. M. (2009a). “Bacteriophages methods and

protocols,” in Methods in Molecular Biology, Vol. 1 (New York, NY: Springer

Nature).

Clokie, M. R. J., and Kropinski, A. M. (2009b). “Bacteriophages methods and

protocols,” in Methods in Molecular Biology, Vol. 2 (New York, NY: Springer

Nature).

Davies, R. L., and Lee, I. (2006). Diversity of temperate bacteriophages

induced in bovine and ovine Mannheimia haemolytica isolates and

identification of a new P2-like phage. FEMS Microbiol. Lett. 260, 162–170.

doi: 10.1111/j.1574-6968.2006.00314.x

Frontiers in Microbiology | www.frontiersin.org 11 April 2018 | Volume 9 | Article 696

Cha et al. Phage Characterization and Therapy for MDR Acinetobacter

Dijkshoorn, L., Nemec, A., and Seifert, H. (2007). An increasing threat in hospitals:

multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5, 939–951.

doi: 10.1038/nrmicro1789

Doi, Y., Murray, G. L., and Peleg, A. Y. (2015).Acinetobacter baumannii: evolution

of antimicrobial resistance-treatment options. Semin. Respir. Crit. Care Med.

36, 85–98. doi: 10.1055/s-0034-1398388

García-Quintanilla, M., Pulido, M. R., López-Rojas, R., Pachón, J., and McConnell,

M. J. (2013). Emerging therapies for multidrug resistant Acinetobacter

baumannii. Trends Microbiol. 21, 157–163. doi: 10.1016/j.tim.2012.12.002

Hua, Y., Luo, T., Yang, Y., Dong, D., Wang, R., Wang, Y., et al. (2018).

Phage therapy as a promising new treatment for lung infection caused

by carbapenem-resistant Acinetobacter baumannii in mice. Front. Microbiol.

8:2659. doi: 10.3389/fmicb.2017.02659

Huang, T. W., Lauderdale, T. L., Liao, T. L., Hsu, M. C., Chang, F. Y., Chang,

S. C., et al. (2015). Effective transfer of a 47 kb NDM-1-positive plasmid

among Acinetobacter species. J. Antimicrob. Chemother. 70, 2734–2738.

doi: 10.1093/jac/dkv191

Huys, I., Pirnay, J.-P., Lavigne, R., Jennes, S., De Vos, D., Casteels, M., et al. (2013).

Paving a regulatory pathway for phage therapy. EMBO Rep. 14, 951–954.

doi: 10.1038/embor.2013.163

Hyman, P., and Abedon, S. T. (2009). Practical methods for determining

phage growth parameters. Methods Mol. Biol. 501, 175–202.

doi: 10.1007/978-1-60327-164-6_18

Jeon, J., D’Souza, R., Pinto, N., Ryu, C. M., Park, J., Yong, D., et al.

(2016a). Characterization and complete genome sequence analysis of two

myoviral bacteriophages infecting clinical carbapenem-resistant Acinetobacter

baumannii isolates. J. Appl. Microbiol. 121, 68–77. doi: 10.1111/jam.

13134

Jeon, J., Ryu, C. M., Lee, J. Y., Park, J. H., Yong, D., and Lee, K. (2016b).

In vivo application of bacteriophage as a potential therapeutic agent to

control OXA-66-Like carbapenemase-producing Acinetobacter baumannii

strains belonging to sequence ype 357.Appl. Environ. Microbiol. 82, 4200–4208.

doi: 10.1128/AEM.00526-16

Keller, B., Dubochet, J., Adrien, M., Maeder, M., Wurtz, M., and Kellenberger,

E. (1988). Length and shape varients of bacteriophage T4 head: mutations I

scaffolding core genes 68 and 22. J. Virol. 62, 2960–2969.

Kim, M. S., Hong, S. S., Park, K., and Myung, H. (2013). Genomic analysis of

bacteriophage PBECO4 infecting Escherichia coli O157:H7. Arch. Virol. 158,

2399–2403. doi: 10.1007/s00705-013-1718-3

Krahn, T., Wibberg, D., Maus, I., Winkler, A., Bontron, S., Sczyrba, A., et al.

(2016). Intraspecies transfer of the chromosomal Acinetobacter baumannii

blaNDM-1 carbapenemase gene.Antimicrob Agents Chemother. 60, 3032–3040.

doi: 10.1128/AAC.00124-16

Kröger, C., Kary, S. C., Schauer, K., and Cameron, A. D. (2017). Genetic regulation

of virulence and antibiotic resistance in Acinetobacter baumannii. Genes 8:12.

doi: 10.3390/genes8010012

Kropinski, A. M., Mazzocco, A., Waddell, T. E., Lingohr, E., and Johnson, R. P.

(2009). Enumeration of bacteriophages by double agar overlay plaque assay.

Methods Mol. Biol. 501, 69–76. doi: 10.1007/978-1-60327-164-6_7

Kusradze, I., Karumidze, N., Rigvava, S., Dvalidze, T., Katsitadze, M.,

Amiranashvili, I., et al. (2016). Characterization and testing the efficiency of

Acinetobacter baumannii phage vB-GEC_Ab-M-G7 as an antibacterial agent.

Front Microbiol. 7:1590. doi: 10.3389/fmicb.2016.01590

Lee, C.-R., Lee, J. H., Park, M., Park, K. S., Bae, I. K., Kim, Y. B., et al.

(2017). Biology of Acinetobacter baumannii: pathogenesis, antibiotic resistance

mechanisms, and prospective treatment options. Front. Cell. Infect. Microbiol.

7:55. doi: 10.3389/fcimb.2017.00055

Li, E., Zhao, J., Ma, Y., Wei, X., Li, H., Lin, W., et al. (2016). Characterization of

a novel Achromobacter xylosoxidans specific siphoviruse: phiAxp-1. Sci. Rep.

6:21943. doi: 10.1038/srep21943

Lin, D. M., Koskella, B., and Lin, H. C. (2017). Phage therapy: an alternative to

antibiotics in the age of multi-drug resistance.World J. Gastrointest. Pharmacol.

Ther. 8, 162–173. doi: 10.4292/wjgpt.v8.i3.162

McConnell, M. J., Actis, L., and Pachon, J. (2013). Acinetobacter baumannii:

human infections, factors contributing to pathogenesis and animal models.

FEMS Microbiol. Rev. 37, 130–155. doi: 10.1111/j.1574-6976.2012.00344.x

Merabishvili, M., Vandenheuvel, D., Kropinski, A. M., Mast, J., De Vos,

D., Verbeken, G., et al. (2014). Characterization of newly isolated lytic

bacteriophages active against Acinetobacter baumannii. PLoS ONE 9:e104853.

doi: 10.1371/journal.pone.0104853

Mihu, M. R., and Martinez, L. R. (2011). Novel therapies for treatment of multi-

drug resistant Acinetobacter baumannii skin infections. Virulence 2, 97–102

doi: 10.4161/viru.2.2.15061

Parasion, S., Kwiatek, M., Gryko, R., Mizak, L., and Malm, A. (2014).

Bacteriophages as an alternative strategy for fighting biofilm development. Pol.

J. Microbiol. 63, 137–145.

Park, K., Cha, K. E., andMyung, H. (2014). Observation of inflammatory responses

in mice orally fed with bacteriophage T7. J. Appl. Microbiol. 117, 627–633.

doi: 10.1111/jam.12565

Regeimbal, J. M., Jacobs, A. C., Corey, B. W., Henry, M. S., Thompson,

M. G., Pavlicek, R. L., et al. (2016). Personalized therapeutic cocktail of

wild environmental phages rescues mice from Acinetobacter baumannii

wound infections. Antimicrob. Agents Chemother. 60, 5806–5816.

doi: 10.1128/AAC.02877-15

Sambrook, J., and Russell, D. W. (2006). Purification of bacteriophage λ particles

by centrifugation through a glycerol step gradient. Cold Spring Harb. Protoc.

2006:pdb.prot3969. doi: 10.1101/pdb.prot3969

Schooley, R. T., Biswas, B., Gill, J. J., Hernandez-Morales, A., Lancaster, J.,

Lessor, L., et al. (2017). Development and use of personalized bacteriophage-

based therapeutic cocktails to treat a patient with a disseminated resistant

Acinetobacter baumannii infection. Antimicrob. Agents Chemother. 61:e00954-

17. doi: 10.1128/AAC.00954-17

Shivaswamy, V. C., Kalasuramath, S. B., Sadanand, C. K., Basavaraju, A.

K., Ginnavaram, V., Bille, S., et al. (2015). Ability of bacteriophage

in resolving wound infection caused by multidrug-resistant Acinetobacter

baumannii in uncontrolled diabetic rats. Microb. Drug Resist. 21, 171–177.

doi: 10.1089/mdr.2014.0120

Tuon, F. F., Rocha, J. L., and Merlini, A. B. (2015). Combined therapy for multi-

drug-resistant Acinetobacter baumannii infection – is there evidence outside

the laboratory? J. Med. Microbiol. 64, 951–959. doi: 10.1099/jmm.0.000144

Turner, D., Wand, M. E., Briers, Y., Lavigne, R., Sutton, J. M., and

Reynolds, D. M. (2017). Characterisation and genome sequence of the

lytic Acinetobacter baumannii bacteriophage vB_AbaS_Loki. PLoS ONE

12:e0172303. doi: 10.1371/journal.pone.0172303

Wang, Y., Mi, Z., Niu, W., An, X., Yuan, X., Liu, H., et al. (2016). Intranasal

treatment with bacteriophage rescues mice from Acinetobacter baumannii-

mediated pneumonia. Fut. Microbiol. 11, 631–641. doi: 10.2217/fmb.16.11

Weber, B. S., Harding, C.M., and Feldman,M. F. (2016). PathogenicAcinetobacter:

from the cell surface to infinity and beyond. J. Bacteriol. 198, 880–887.

doi: 10.1128/JB.00906-15

Yang, H., Liang, L., Lin, S., and Jia, S. (2010). Isolation and characterization of

a virulent bacteriophage AB1 of Acinetobacter baumannii. BMC Microbiol.

10:131. doi: 10.1186/1471-2180-10-131

Zhang, G., Leclercq, S. O., Tian, J., Wang, C., Yahara, K., Ai, G., et al. (2017).

A new subclass of intrinsic aminoglycoside nucleotidyltransferases, ANT(3")-

II, is horizontally transferred among Acinetobacter spp. by homologous

recombination. PLoS Genet. 13:e1006602. doi: 10.1371/journal.pgen.1006602

Zhang, J., Liu, X., Li, and Li, X. J. (2015). Bioinformatic analysis of phage AB3,

a phiKMV-like virus infecting Acinetobacter baumannii. Genet. Mol. Res. 14,

190–198. doi: 10.4238/2015

Conflict of Interest Statement: The authors declare that the research was

conducted in the absence of any commercial or financial relationships that could

be construed as a potential conflict of interest.

Copyright © 2018 Cha, Oh, Jang, Jo, Kim, Ha, Ko andMyung. This is an open-access

article distributed under the terms of the Creative Commons Attribution License (CC

BY). The use, distribution or reproduction in other forums is permitted, provided

the original author(s) and the copyright owner are credited and that the original

publication in this journal is cited, in accordance with accepted academic practice.

No use, distribution or reproduction is permitted which does not comply with these

terms.

Frontiers in Microbiology | www.frontiersin.org 12 April 2018 | Volume 9 | Article 696